Multiplexed Detection of Protein−Peptide Interaction and Inhibition Using Capillary Electrophoresis

Yang, Peilin; Whelan, Rebecca J.; Mao, Ying; Lee, Angel W.-M.; Carter‐Su, Christin; Kennedy, Robert T.

doi:10.1021/ac061936e

Cited by 40 publications

(38 citation statements)

References 38 publications

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“…In APCE, an equilibrated mixture of binding partners is electrophoresed to allow for detection of non-covalent interactions. 1–3 APCE has been used to investigate many biomolecular interactions such as protein-protein 4–7 , antibody-antigen 8–13 , protein-DNA 10,14–17 , protein-peptide 10,18 and protein-aptamer 19–21 . Typically, one binding partner is fluorescently labeled enabling sensitive detection by laser induced fluorescence (LIF).…”

Section: Introductionmentioning

confidence: 99%

Protein Cross-Linking Capillary Electrophoresis for Protein–Protein Interaction Analysis

et al. 2016

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Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying and screening for modulators of protein-protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then the complex separated from free protein allowing direct determination of bound to free ratios. Although possessing many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain non-covalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS-complexes. The method gives good quantitative results, e.g., a lysozyme-antibody interaction was found to have Kd = 24 ± 3 nM by PXCE and Kd = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have Kd = 25 ± 5 nM by PXCE which agrees with Kd values reported without cross-linking. Hsp70-Bag3 binding site mutants and small molecule inhibitors of Hsp70-Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC50 values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs.

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Section: Introductionmentioning

confidence: 99%

Protein Cross-Linking Capillary Electrophoresis for Protein–Protein Interaction Analysis

et al. 2016

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“…[69,70] A four-plexed assay of protein kinases demonstrated simultaneous resolution of distinct substrates and products. [69] A study of protein-peptide interaction targets with src homology 2 (SH2) domains simultaneously assayed three proteins (Figure 4).…”

Section: Improvements To Throughputmentioning

confidence: 99%

Advances in capillary electrophoresis and the implications for drug discovery

Ouimet

D'Amico

Kennedy

2016

Expert Opinion on Drug Discovery

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Introduction Many screening platforms are prone to assay interferences that can be avoided by directly measuring the target or enzymatic product. Capillary electrophoresis (CE) and microchip electrophoresis (MCE) have been applied in a variety of formats to drug discovery. CE provides direct detection of the product allowing for the identification of some forms of assay interference. The high efficiency, rapid separations, and low volume requirements make CE amenable to drug discovery. Areas Covered This article describes advances in capillary electrophoresis throughput, sample introduction, and target assays as they pertain to drug discovery and screening. Instrumental advances discussed include integrated droplet microfluidics platforms and multiplexed arrays. Applications of CE to assays of diverse drug discovery targets, including enzymes and affinity interactions are also described. Expert opinion Current screening with CE does not fully take advantage of the throughputs or low sample volumes possible with CE and is most suitable as a secondary screening method or for screens that are inaccessible with more common platforms. With further development, droplet microfluidics coupled to MCE could take advantage of the low sample requirements by performing assays on the nanoliter scale at high throughput.

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“…CE and hyphenated CE techniques, in particular, have been employed given their high separation efficiency, small sample requirements, and reproducibility [31]. Furthermore, CE technology demonstrates high sensitivity, relatively short analysis times, and the potential for parallel operation leading to highthroughput screening capabilities [32][33][34]. The latter characteristics are of principal importance considering the need for expanded time-and dose-dependent study of MT induction.…”

Section: Model Applications and Experimental Conditionsmentioning

confidence: 99%

Biologically driven neural platform invoking parallel electrophoretic separation and urinary metabolite screening

et al. 2012

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This work reveals a computational framework for parallel electrophoretic separation of complex biological macromolecules and model urinary metabolites. More specifically, the implementation of a particle swarm optimization (PSO) algorithm on a neural network platform for multiparameter optimization of multiplexed 24-capillary electrophoresis technology with UV detection is highlighted. Two experimental systems were examined: (1) separation of purified rabbit metallothioneins and (2) separation of model toluene urinary metabolites and selected organic acids. Results proved superior to the use of neural networks employing standard back propagation when examining training error, fitting response, and predictive abilities. Simulation runs were obtained as a result of metaheuristic examination of the global search space with experimental responses in good agreement with predicted values. Full separation of selected analytes was realized after employing optimal model conditions. This framework provides guidance for the application of metaheuristic computational tools to aid in future studies involving parallel chemical separation and screening. Adaptable pseudo-code is provided to enable users of varied software packages and modeling framework to implement the PSO algorithm for their desired use.

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Multiplexed Detection of Protein−Peptide Interaction and Inhibition Using Capillary Electrophoresis

Cited by 40 publications

References 38 publications

Protein Cross-Linking Capillary Electrophoresis for Protein–Protein Interaction Analysis

Protein Cross-Linking Capillary Electrophoresis for Protein–Protein Interaction Analysis

Advances in capillary electrophoresis and the implications for drug discovery

Biologically driven neural platform invoking parallel electrophoretic separation and urinary metabolite screening

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